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Simulation Study for the Degradation of Some Insecticides during
Different Black Table Olive Processes
Aysegul Yildirim Kumral,* Nabi Alper Kumral, Aysenur Kolcu, Busra Maden, and Buse Artik
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ABSTRACT: The aim of this study was to determine the effects of different olive
processing methods on deltamethrin (DEL), dimethoate (DIM), and imidacloprid
(IMI), the most commonly preferred synthetic insecticides for controlling olive
pests such as the olive fruit fly. The hypothesis is that the fermentation could
accelerate the degradation process of the insecticides. For this purpose, olives were
left for fermentation (natural black olives) without and with starter addition (two
Lactobacillus plantarum strains 112 and 123) and processed as dehydrated black
olives. To monitor the degradation rate of insecticides, olives were first polluted
with the insecticides and then the residues were detected periodically during the
processes. The insecticide degradation rates were found to be significantly higher in natural black olives and natural black olives
inoculated with both starters compared with those of crude olives and dehydrated black olives. At the end of fermentation (after 60
d), 53−61% of deltamethrin, 66−68% of dimethoate, and 42−50% of imidacloprid were removed in natural black olives and natural
black olives inoculated with both starters. In dehydrated olives, the degradation of deltamethrin, dimethoate, and imidacloprid was
lower with rates of 9.7, 40, and 13.4%, respectively. The current study demonstrated that natural and starter-added natural black
olive processing accelerated the degradation of deltamethrin, dimethoate, and imidacloprid.
1. INTRODUCTION DEL is oral LD50 of 114−168 mg/kg for rats. A NEO
Pesticides are one of the major inputs used for increasing the substance, IMI was first used in 1991 as a systemic
yield of agricultural commodities. Nevertheless, the presence of acetylcholine receptor agonist insecticide with contact and
pesticide residues on processed food products is a crucial stomach action. The compound is moderately toxic, having an
problem causing safety and health problems.1 Table olives have acute oral LD50 of 131 mg/kg for rats and causing side effects
7
great economic importance especially for the Mediterranean on the reproduction and development in humans. The half-
countries and other olive-producing areas because of high lives of DIM, DEL, and IMI are 7.2−15.5, 11−19, and 174−
production and consumption rates. The intensive use of 191 days, respectively, depending on the hydrolysis activities
insecticides for the main pests of olive trees leads to increased and metabolism.8−10
residues on olive fruits.2 Although alternative control methods Pesticides can be degraded by photolysis, hydrolysis,
have been implemented in many countries, unfortunately the oxidation and reduction, and metabolism (plants, animals, or
broad-spectrum synthetic insecticides, organophosphorus microorganisms) and affected by temperature and pH.
(OPs), synthetic pyrethroids (SPs), and neonicotinoids Different food processing and preservation techniques,
(NEOs) are still the most commonly preferred insecticides postharvest treatments, and cold storage have also been
for the control of the pests.3−5 However, improper use of these found to be effective. Techniques based on concentration
compounds can cause residue problems on agricultural (drying/dehydration and concentration) increased the pesti-
products if the necessary precautions are not taken. cide residue levels in the end products, whereas milling, baking,
Deltamethrin (DEL), dimethoate (DIM), and imidacloprid winemaking, malting, and brewing lowered their levels in these.
(IMI) are the most common substances used during olive
6 Refining, fermentation, and curing have been reported to affectgrowing. Although numerous OP substances have been the pesticide level in foods to a varied extent.11
restricted in European Union countries, the use of DIM is
still permitted in large parts of the world.4,5 DIM has been
registered since 1951 as a systemic acaricide and insecticide Received: April 25, 2020
with contact and stomach action. The compound is moderately Accepted: May 19, 2020
toxic with an acute oral LD50 of 245 mg/kg for rats and a
Published: June 1, 2020
relative risk as a cholinesterase inhibitor in humans. A SP
compound, DEL has been widely used since the 1980s on
various crops and human-disease vectors. The acute toxicity of
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Fermentation is a microbiological process in which enzymes
transform carbohydrates, typically starch or sugar, into simpler
components such as alcohols, acids, and gases and most of the
proteins to amino acids and low-molecular-weight peptides.
The most common groups of microorganisms involved in the
fermentation of food, include yeasts, bacteria, and moulds,
which produce enzymes that catalyze the fermentation
process.12 The biological degradation of the pesticides by
microorganisms is dependent on the structure of the chemical
(volatility, insolubility in water, and adsorption ability to matrix
compounds) and some environmental parameters (temper-
ature, pH, moisture, and light).13
Some lactic acid bacteria (LAB), belonging to Lactobacillus
and Leuconostoc genera, can metabolize broad-spectrum
synthetic insecticides, i.e., OPs, SPs, NEOs, by their esterase
enzymes and/or using the insecticides as carbon and energy
sources.14−21 Besides, LAB have gained a lot of interest due to
their health benefits and are widely used as probiotics and
starter cultures for fermented products because of their
generally recognized as safe (GRAS) status.22 Biodegradation
of pesticides is a promising technology because of its potential
for the removal of residues from food and agricultural
products.13
There are several trade preparations for table olives such as
alkali-treated olives, natural olives, dehydrated/shrivelled
olives, and olives darkened by oxidation.23,24 There is no
scientific information about the effect of different handling and
processing methods on pesticide residues in olives. This
research was focused on natural black olives and dehydrated
black olives techniques because of the increasing demand of Figure 1. Effects on degradation of imidiacloprid (IMI) in olive fruits
consumers to chemical-free, natural, and minimally processed processed by different methods: crude olives (CO, control);
food products. Natural black olives (NBOs) and dehydrated dehydrated and/or shrivelled black olives (DBO); natural black
black olives (DBOs) are popular black table olive types. NBOs olives (NBO); natural black olives with Lactobacillus plantarum 112
are obtained by placing fruits directly in brine with 8−9% salt, (NBO112); and natural black olives with L. plantarum 123 with L.
where they undergo complete or partial fermentation, and plantarum 123 (NBO123).
preserved or not by the addition of acidifying agents.24
Traditional NBO production is a spontaneous fermentation day 60. But, in CO and DBO, a significant decrease in IMI
process that relies upon microorganisms present in fresh fruits amounts was found on day 60 (F4,4 = 86.2, P < 0.01).
and the processing environment. It is reported that the use of Nevertheless, these degradation rates in both CO and DBO
suitable starter cultures in NBO processing may help to did not reach the rates of all NBO treatments. In addition, LAB
standardize the fermentation, improve the microbiological inoculation into NBO did not affect the IMI amount (F16,16 =
quality, and increase the lactic acid yield to provide table olives
25−28 29.2, P < 0.01) (Figure 1).of higher quality. On the other hand, DBOs are generally
24 Eight and 30 days after pollution with DEL, the insecticideobtained by partial dehydration in coarse salt. amount was significantly decreased in NBO, NBO112, and
The aim of this simulation study was to compare the effects NBO123 (11−22% on day 8 and 54−57% on day 30) (F4,4 =
of different olive processing methods (DBO, NBO) and LAB 82.9, P < 0.01). On days 8, 30, and 60, the DEL amounts in
[NBO inoculated with lactic starter (NBOS)] on insecticide NBO, NBO112, and NBO123 treatments were significantly
residues in the table olives polluted with DIM, DEL, and IMI. lower compared to those in both CO and DBO treatment (F4,4
= 180.2, P < 0.01). The reduction rates in NBO112 and
2. RESULTS NBO123 treatments were higher than those in NBO from day
2.1. Insecticide Residues. Changes in the IMI, DEL, and 8 to 30 (F16,16 = 13.4, P < 0.01) (Figure 2).
DIM levels in crude olives (CO, control), DBO, NBO, NBO A significant amount of DIM was decomposed rapidly in
with L. plantarum 112 (NBO112) and NBO with L. plantarum NBO, NBO112, and NBO123 treatments after 8 days (32−
123 (NBO123) are demonstrated in Figures 1−3. Eight days 37% reduction). After day 8, the reduction rates in NBO were
after the pollution with IMI, the insecticide amount was significantly higher compared with those in CO and DBO
significantly decreased in NBO, NBO112, and NBO123 treatment. The differences among DIM amounts in NBO,
treatments (54, 51, and 61% reduction, respectively, F4,4 = NBO112, and NBO123 treatments were not significant (F4,4 =
335.26, P < 0.01). The degradation rates in DBO were not 197.9, P < 0.01). On day 60, a significantly large portion of the
significantly different from those in CO. The highest IMI DIM amount (66−68%) was degraded in all NBO treatments
residue was detected in CO, followed by DBO on day 8. A (F4,4 = 171.2, P < 0.01). However, time−treatment interactions
significant lower IMI residue was measured in NBO112 on the were found to be significant. Residues in CO and DBO at
same detection day. In NBO, NBO112, and NBO123 different time intervals were higher than those in all NBO
treatments, there was no significant change from day 8 to treatments (F16,16 = 17.8, P < 0.01). Addition of starters (both
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Figure 3. Effects on degradation of dimethoate (DIM) in olive fruits
processed by different methods: crude olives (CO, control);
Figure 2. Effects on degradation of deltamethrin (DEL) in olive fruits dehydrated and/or shrivelled black olives (DBO); natural black
processed by different methods: crude olives (CO, control); olives (NBO); natural black olives with L. plantarum 112 (NBO112);
dehydrated and/or shrivelled black olives (DBO); natural black and natural black olives with L. plantarum 123 (NBO123).
olives (NBO); natural black olives with L. plantarum 112 (NBO112);
and natural black olives with L. plantarum 123 (NBO123).
L. plantarum 112 and 123) to NBOs was not affected the DIM of the yeast cells was not affected by the starter addition in all
residues (Figure 3). treatments with DIM (DIM F2,2 = 1.97, P = 0.16). The yeast
2.2. Microbiological Changes. The growth of mesophilic numbers were significantly varied depending on the observa-
aerobic bacteria (MAB), yeasts and moulds (YM), enter- tion time (DEL F4,4 = 114.38, P < 0.01; DIM F4,4 = 26.62, P <
obacteria (ENB) and LAB during NBO treatments in the 0.01, IMI F4,4 = 7.27, P < 0.01) in all trials. Although the
presence of DEL, DIM, and IMI are shown in Figure 4. ENB highest yeast numbers were seen on the 15th day in all trials,
growth was not detected in all treatments. For all insecticide no significant difference was detected between the treatments
trials, a significant LAB growth (Figure 4) was detected in on this day.
NBO treatments, which were inoculated with starters The growth of total MAB showed differences between NBO
(NBO112 and NBO123), compared with their corresponding and NBOs with the addition of starter (Figure 4). Based on the
spontaneously fermented NBO treatments (Figure 4; DEL F2,2 MANOVA test, the addition of both starters significantly
= 700.49, P < 0.01; DIM F2,2 = 870.8, P < 0.01, IMI F2,2 = increased the number of MAB cells (DEL F2,2 = 197.49, P <
741.11, P < 0.01). The initial LAB concentration was between 0.01; DIM F2,2 = 189.53, P < 0.01, IMI F2,2 = 125.82, P <
6.89 and 7.86 cfu/mL, and there was a continuous LAB 0.01). The cell numbers in different observation times were
existence during the whole process. No significant difference found to be significantly different (DEL F4,4 = 31.83, P < 0.01;
was detected between NBO123 and NBO112 trials for all DIM F4,4 = 10.19, P < 0.01, IMI F4,4 = 7.43, P < 0.01) in all
insecticides. The growth of LAB was decreased significantly treatments. Time−treatment interactions were found to be
from day 0 to 7, but no difference was found between days 7 significant in all treatments (DEL F8,8 = 16.44, P < 0.01; DIM
and 60, except for the NBO123 treatments of DIM and IMI F8,8 = 4.17, P < 0.01, IMI F8,8 = 2.73, P = 0.02).
(DEL F4,4 = 4.74, P < 0.01; DIM F4,4 = 10.36, P < 0.01, IMI 2.3. pH Changes. The pH changes of NBO brines are
F4,4 = 11.02, P < 0.01). demonstrated in Figure 5. Marked differences were observed
There was evident yeast growth in all treatments during between the NBO and the starter-inoculated NBO in all
trials (Figure 4), with initial cell numbers between 1.84 and pesticide-containing treatments. The pH decline (below 4) was
2.93 log cfu/mL. The yeast growth displayed fluctuations; significant in all starter-added treatments. In NBOs polluted
however, at the end of 60 days, the number of yeasts reached with DEL and DIM, the pH was constantly higher than 4
higher levels (2.30−4.79 log cfu/mL) than their initial during the whole process, but in NBOs polluted with IM, the
numbers in all treatments, except in NBO112 polluted with pH started to decrease after the 15th day and finally reached
IMI. The results of the MANOVA test showed that the growth below pH 4.
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Figure 4. Mean microbial growth [mesophilic aerobic bacteria (MAB), lactic acid bacteria (LAB), yeast and moulds (YM)] in the presence of
imidiacloprid (IMI), deltamethrin (DEL), and dimethoate (DIM) during different olive processing methods: natural black olives (NBO); natural
black olives with L. plantarum 112 (NBO112); and natural black olives with L. plantarum 123 (NBO123).
Figure 5. pH changes in the presence of imidiacloprid (IMI), deltamethrin (DEL), and dimethoate (DIM) during different olive processing
methods: natural black olives (NBO); natural black olives with L. plantarum 112 (NBO112); and natural black olives with L. plantarum 123
(NBO123).
3. DISCUSSION those in DBO and CO. When olive fruits were polluted with
In the present study, it has been demonstrated that insecticide DEL, DIM, and IMI, they were degraded by 11−22%, 32−
degradation rates were found to be significantly higher in 37%, and 51−61% after a fermentation period of 8 days and
NBO, NBO112, and NBO123 treatments compared with 53−61%, 66−68%, and 42−50% after a fermentation period of
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60 days (optimum fermentation time), respectively, in NBO, compared with those without inoculation (NBO), the presence
NBO112, and NBO123 treatments. In accordance with our of L. plantarum 112 and L. plantarum 123 did not affect the
results, some researchers reported that fermentation generally insecticide degradation. Additionally, the trend of yeast cell
causes a decrease in OPs and SPs levels in processed foods growth was related to insecticide degradation. The yeast cell
such as cider, vinegar, kimchi (fermented Chinese cabbage), growth increased during the first 15 days, when the insecticides
wheat, and skimmed milk.14,16,22,29−31 Similarly, DIM and were degraded rapidly. In a research by Randazzo et al. on
DEL were degraded during the fermentations of wine, bread, fermented green olives, LAB and yeast populations were
and yoghurt.32−34 It has been previously demonstrated that affected by the presence of copper.50 The yeast growth was
some OPs and SPs, namely, chlorpyrifos, malathion, detected at the beginning of fermentation and was constant till
methidathion, parathion, dimethoate, bifenthrin, deltametrin, the end of the process. This was in accordance with results of
permethrin, and fenvalerate, degraded during the fermentation Ohshiro et al., who reported that the degradative role of the
of some foods containing LAB and yeasts.16,22,34−37 Kawar et microorganisms accelerates in association with yeasts present
al. showed that methidathion (OPs) and dimethoate levels in the medium.51
were degraded about 46 and 85%, respectively, in fermented
wine after a fermentation period of 57 days.35 Moreover, 4. CONCLUSIONS
Banna and Kawar demonstrated that the parathion (OPs)
levels in cider and vinegar decreased about 70 and 80%, after a The degradation rates of three different insecticides did not
fermentation period of 12 and 57 days, respectively.29 exceed 14% after 30 days in dry salted olive process. In
Fatichenti et al. found that some SPs (deltamethrin, addition, the insecticide residues in the dry salted olives were
permethrin, and fenvalerate) were almost totally degraded similar to those in the crude olive samples stored in the dark at
with yeast (Saccharomyces cerevisiae) activity after a fermenta- room temperature. This can lead to a risk of high chemical
tion period of 9 days.36 Cho et al. reported that chlorpyrifos residues during the consumption of the product. More than
was degraded quickly with LAB activity within 3 days (83.3%) half of the chemical residues were degraded after 30 days in
during Kimchi fermentation.16 olives polluted with three insecticides and processed in brine
On the other hand, our study demonstrated that the under the same conditions, despite the insecticides having
degradation rates in DBO were 1.5, 3.8, and 0% after 8 days different chemical structures. This shows that the brining
and 14, 1.5, and 0% after 30 days (optimum consumption time process in black olives is a useful method for the reduction of
for DBO), respectively, for DEL, DIM, and IMI. These insecticide residues. In fact, even if the LAB is inoculated
unfavorable insecticide residues and their stabilities in DBO artificially to the brines, the effect of the inoculation on the
processing may be caused by water loss during the dehydration pesticide degradation often does not change much.
of olives under dark-room conditions.38 El Beit et al. revealed This research has shown that the consumption of dry salted
that the pesticide levels did not change under acid conditions olives may cause a great risk when no attention is paid to
or high salt concentrations.38 preharvest intervals and good agricultural practices. Thefermentation is generally used for improving the nutritional
Pesticide degradations are dependent on several processing quality and shelf life of foods, in addition to its positive effects
and environmental conditions such as temperature, light,
39−41 on the decontamination of chemical contaminants, such asmoisture, and pH. In general, OP, SP, and NEO pesticides. Although black olive brining accelerates the
insecticides are stable in acidic pH and easily degraded in
38,42−44 degradation of the insecticides, this process, in which there isalkali pH. Some researchers found that IMI was slowly no ultraviolet light from the sun, is not fully successful in
hydrolyzed and was stable between pH 4 and 9 when protected removing all insecticide residues by the end of fermentation.
from light under sterile conditions. Hydrolysis was more rapid Therefore, good agricultural practices in olive orchards should
under alkaline (up to pH 9) conditions.42,44 Generally, the pH be the first priority.
becomes lower (≤4) during olive fermentations, which is
similar to our results (Figure 5). Previous soil studies showed
that the reason of insecticide degradation is both chemical 5. EXPERIMENTAL SECTION
hydrolysis in alkaline pH and microbial activity.39,45,46 5.1. Chemicals and Reagents. The analytical standard
Therefore, the degradation in the present study could be reagents, dimethoate [DIM; O,O-diethyl-O-3,5,6-trichloro-2-
caused by microbial activity rather than hydrolysis due to the pyridyl phosphorothioate], deltamethrin [DEL; (S)-cyano-(3-
acidic conditions in the medium. It was reported that some phenoxyphenyl)methyl] (1R,3R)-3-(2,2-dibromoethenyl)-2,2-
microbial agents can metabolize insecticides by their esterase dimethylcyclopropane-1-carboxylate], and imidiacloprid [IMI;
enzymes and using these compounds as carbon and energy (E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylide-
sources.39,47 It is well known that some bacteria can metabolize neamine], were purchased from Dr. Ehrenstorfer GmbH
insecticides by their specific enzymes such as esterase.48 (Germany). Emulsifiable concentrate commercial substances
Several studies have previously shown that many OPs, DIM (40 g/L Poligor, Hektas ̧ Company, Turkey), DEL (25 g/
including esters of phosphoric acid, could be hydrolyzed by L Deltharin, Hektas ̧ Company, Turkey), and IMI (350 g/L
carboxylesterase and phosphotriesterase.11,18 In addition, some Confidor, Bayer Company, Germany) were also obtained from
microbial agents (e.g., S. cerevisiae, L. plantarum, Lactobacillus manufacturers. All other reagents were analytical grade. Salts
bulgaricus, Lactobacillus paracasei, Leuconostoc mesenteroides, used during olive processing were obtained from local markets.
Lactobacillus brevis, Lactobacillus sakei, and Lactobacillus casei) 5.2. Olive Fruits. Olive fruits of “Gemlik” cv. were
used OPs and SPs insecticides as carbon and energy sources in harvested at the black-ripe stage suitable for NBO and DBO
some processed food media.15−17,20,21,49 In the current study, productions from an experimental olive orchard in Orhangazi
activities of yeasts and bacteria were determined in NBO district of Bursa, Turkey, in 2018. None of the pesticides were
treatments. Although significant LAB cell growth was found in applied to olive trees in this orchard during the growing
black table olives inoculated with the two L. plantarum strains season, and the olive fruits were free from all pesticides.
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5.3. Starter Microorganisms. Two L. plantarum strains
(112 and 123) used in this research were previously isolated
from the fermentation brines of NBO. Strains were identified
by 16s rRNA technique and differentiated from other group
members according to Torriani et al.52,53 L. plantarum strains
were propagated in De Man, Rogosa, and Sharpe (MRS) broth
at 30 °C. Eighteen to 24 h old test strains at a concentration of
108−109 cfu/mL were centrifuged at 10 000g, washed twice in
sterile saline, and resuspended in brine.15 NBOSs were
inoculated with strains at a final concentration of 107−108
cfu/mL.
5.4. Insecticide Pollution of Table Olives and
Experimental Design. Crude olives of all treatments were
homogeneously sprayed with commercial formulations of
DIM, DEL, and IMI, separately, using a Potter precision
spray tower at the 10 bar and 3 s settings (Burkard
Manufacturing Co. Ltd., Rickmansworth, UK). The fruits
were polluted at the following doses (DEL: 14 mg/L, DIM: 8.5
mg/L, and IMI: 7 mg/L), which are quite above the Maximum
Residue Limits (MRL, DEL: 1.0 mg/L, DIM: 3.0 mg/L, and
IMI: 0.5 mg/L) for olive products in European countries.5 The
insecticide degradation changes were investigated by five
different treatments: (i) CO, (ii) DBO, (iii) NBO, (iv)
NBO112 and (v) NBO123. All of the treatments and analyses
were done in triplicate, and all jars and bottles were kept at
room temperature (Table 1).
5.5. Monitoring the Growth of Test Strains. Brine
samples were microbiologically analyzed at regular intervals for
MAB, LAB, ENB, and YM.54,55 Enumeration of micro-
organisms was carried out using a spiral plating system (Easy
Spiral, Interscience, France). Appropriate dilutions were plated
on Plate Count Agar, MRS Agar, and Oxytetracycline Glucose
Yeast Extract Agar (Merck KGaA, Darmstadt, Germany) for
MAB, LAB, and YM, respectively, and incubated at 30 °C for
48 h. Violet Red Bile Glucose Agar (Merck KGaA, Darmstadt,
Germany) was used for the enumeration of ENB and
incubated at 35 °C for 48 h. The curves of cell growth for
each microbial group were plotted using Excel program of
Windows.
5.6. Monitoring the pH. The pH of all NBO treatments
was monitored periodically in brines with a pH 315i model
(WTW, Germany) pHmeter.
5.7. Insecticide Extraction Procedure. Samples from all
experiments (60 g) were crushed and homogenized, and
aliquots of 15 g of each were used for extraction and analysis
for pesticides. Extraction and partition of insecticides were
done with the Quick, Easy, Cheap, Effective, Rugged, and Safe
(QuEChERS) method based on the manufacturer’s instruc-
tions.56 Briefly, each homogenized olive sample was put in a 50
mL polypropylene centrifuge tube and then added with 10 mL
of acetonitrile (containing 1% acetic acid). The tubes were
hand-shaken vigorously for 1 min. Then, the tubes were added
with 6 g of anhydrous MgSO4 and 1.5 g of sodium acetate
(NaAc), shaken vigorously for 1 min, and centrifuged at 5000
rpm for 2 min. After that, 8 mL of the supernatant was
collected from each tube and transferred into a 15 mL falcon
tube containing 1200 mg of anhydrous MgSO4, 400 mg of
PSA, and 400 mg of C18. The tubes were mixed with a vortex
for 30 sn and then centrifuged at 5000 rpm for 2 min. Lastly, 1
mL of supernatant from each tube was transferred into glass
autosampler vials for further liquid chromatography with
tandem mass spectrometry (LC-MS-MS) analysis.
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Table 1. Experimental Design, Treatments, and Conditions
sample
insecticides sample name code treatments & conditions
for each insecticide crude olives, Control CO 60 g of crude olives were sprayed with insecticides, put into 105 mL volume glass jars without any treatment, and kept at room temperature
(DIM, DEL, and IMI) dehydrated black olives DBO 60 g of crude olives were sprayed with insecticides, put in 190 mL volume glass jars with 12 g of coarse salt (20%), mixed effectually, and kept at room
temperature
natural black olives NBO 60 g of crude olives were sprayed with insecticides, put into 105 mL volume glass jars in brines containing 6% salt immediately after harvest, and allowed to
ferment spontaneously at room temperature
natural black olives with NBO112 60 g of crude olives were sprayed with insecticides, put into 105 mL volume glass jars in brines containing 6% salt immediately after harvest, inoculated with
L. plantarum 112 L. plantarum 112 at a final concentration of 107−108 cfu/mL, and allowed to ferment at room temperature
natural black olives with NBO123 60 g of crude olives were sprayed with insecticides, put into 105 mL volume glass jars in brines containing 6% salt immediately after harvest, inoculated with
L. plantarum 123 L. plantarum 123 at a final concentration of 107−108 cfu/mL, and allowed to ferment at room temperature
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5.8. LC-MS-MS Analysis. Concentrations of DIM, DEL, OUAP(Z)-2015/9. Authors also thank Gulden Hazarhun for
and IMI were measured using Agilent Technologies 6470 technical assistance during pesticide analyses. There is no
Triple Quad Liquid-Mass Spectrometry (Agilent, Santa Clara, conflict of interest about this research. Aysegul Yildirim
CA). Choromatographic separation was achieved by gradient Kumral designed and set up the experiments, Nabi Alper
elution using an Agilent Poroshell SB-C18 column (3 × 100 Kumral performed insecticide pollution and the data analyses,
mm × 2.7 μm). One microliter of filtrate was injected into the Aysegul Yildirim Kumral, Busra Maden, and Buse Artik
LC-MS-MS. The mobile phase consisted of A, water at 0.1% performed the microbial tests, Nabi Alper Kumral and Aysenur
formic acid with 1 mM of ammonium formate, and B, Kolcu made pesticide analysis, and all authors contributed in
methanol. The gradient program was as follows: 0−0.5 min writing, reading, and approving the manuscript.
70% A, 0.5−8 min 70% A, 8−12.5 min 5% A, 12.5−12.6 min
5% A, and 12.6−15.0 min 70% A. The mobile phase flow rate ■ REFERENCES
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− pesticide residues in food - a review. J. Food Sci. Technol. 2014, 51,Aysegul Yildirim Kumral Department of Food Engineering, 201−220.
Faculty of Agriculture, Bursa Uludag University, 16059 Bursa, (12) Paredes-Loṕez, O.; Gonzalez-Casteneda, J.; Carabenz-Trejo, A.
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of Agriculture, Bursa Uludag University, 16059 Bursa, Turkey effects on fermentation and food quality. Crit. Rev. Food Sci. Nutr.
Aysenur Kolcu − Department of Plant Protection, Faculty of 2015, 55, 839−863.
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Busra Maden − Department of Food Engineering, Faculty of heterologous gene expression of a novel esterase from Lactobacillus
Agriculture, Bursa Uludag University, 16059 Bursa, Turkey casei CL96. Appl. Environ. Microbiol. 2004, 70, 3213−3221.
Buse Artik − Department of Food Engineering, Faculty of (15) Cho, K. M.; Math, R. K.; Islam, S. M.; Lim, W. J.; Hong, S. Y.;
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